Chapter 75

gp130 Activation in Müller Cells is Not Essential for Photoreceptor Protection from Light Damage

Yumi Ueki, Srinivas Chollangi, Yun-Zheng Le, and John D. Ash

Abstract Members of IL-6 family cytokines, such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), activate the common signaltransducing receptor gp130. We and others have previously shown that application of exogenous gp130 ligands promotes photoreceptor survival in light-induced and inherited retinal degeneration in animal models. While there is strong evidence that gp130 plays an essential role in photoreceptor protection, it is not clear whether protection is cell-autonomous in photoreceptors or an effect of Müller cell activation. To investigate the role of Müller cells in gp130-mediated photoreceptor protection, we have generated conditional gp130 knockout (KO) mice in retinal Müller cells using the Cre/lox system. Western blot and immunohistochemical analyses show that in our conditional gp130 KO mice, approximately 50% Müller cells no longer respond to LIF with activation of known downstream signaling proteins, STAT3 and ERK1/2. Despite the loss of gp130 activity in many Müller cells, intravitreal injection of LIF still induced significant degree of photoreceptor protection that was comparable to normal littermates. These data suggest that Müller cell activation of gp130 is not essential for photoreceptor protection, and support the hypothesis that the protection is mediated by cell-autonomous mechanisms in photoreceptors.

75.1 Introduction

Signal-transducing receptor gp130 is a common receptor for interleukin (IL)-6 family of cytokines, such as ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and cardiotropin-like cytokine (CLC). Application of exogenous gp130 ligands has been shown to promote photoreceptor survival in both light-induced and

Y. Ueki (B)

Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_75,C Springer Science+Business Media, LLC 2010

inherited models of retinal degeneration (LaVail et al. 1992; Cayouette et al. 1998; LaVail et al. 1998; Chong et al. 1999; Liang et al. 2001; Bok et al. 2002; Song et al. 2003).

We have shown that activation of gp130 by intravitreal injection of LIF preserves photoreceptor function and prevents photoreceptor cell death against light-induced oxidative stress (light damage) in dose-dependent manner (Ueki et al. 2008). While there is strong evidence that gp130 plays an essential role in survival of photoreceptors, the mechanism by which this is accomplished is not well established.

Activated gp130 signals through the Jak/STAT3, ERK1/2, and PI3K/Akt pathways (Boulton et al. 1994; Heinrich et al. 2003; Oh et al. 1998). In the rodent retina, activation of STAT3 and/or ERK1/2 is detected in Müller cells shortly after the injection of gp130 ligands (Wahlin et al. 2000; Peterson et al. 2000; Wen et al. 2006), suggesting that Müller cell activation of gp130 is essential for photoreceptor protection. On the other hand, our previous study has demonstrated that all retinal cells, including photoreceptors activate STAT3 at the time of light damage (Ueki et al. 2008), suggesting photoreceptor protection requires direct gp130 activation in photoreceptors. Thus, it is not known whether protection is cell autonomous in photoreceptors or an effect of Müller cell activation. The purpose of this study was to investigate whether photoreceptor protection by gp130 ligands requires activation of gp130 in Müller cells. To accomplish this, we generated conditional gp130 knockout mice in Müller cells using the Cre/lox system.

75.2 Conditional gp130 Knockout in the Retinal Müller Cells

In order to study Müller cell-specific roles of gp130 in photoreceptor protection, we have generated conditional gp130 knockout (KO) mice using the Cre/lox system. We have mated mice homozygous for floxed gp130 allele (gp130f/f) (Betz et al. 1998) with VMD2-cre transgenic line (Ueki et al. 2009). gp130f/f/VMD2-cre+ mice were viable and had no apparent phenotypic abnormalities. All mice used for experiments were albino and 6- to 7-week old.

To assess the extent of cell-specific gp130 deletion in the retina, intravitreal injection of a gp130 ligand, leukemia inhibitory factor (LIF) was performed, and activation of known downstream signaling proteins was detected by immunohistochemistry (IHC) (Fig. 75.1). Our previous study has shown that injection of LIF can induce robust STAT3 and ERK1/2 activation in Müller cell 30 min after the injection (Ueki et al. 2008). As a result of Cre-mediated recombination of gp130, STAT3 and ERK1/2 activation was significantly reduced in gp130f/f/VMD2cre+mice 30 min after the injection and the loss was localized to Müller cells by IHC (Fig. 75.1; ERK1/2 data not shown). Our data show that approximately 50% Müller cells in gp130f/f/VMD2-cre+ retinas do not respond to gp130 ligands as a result of Cre-mediated knockout of gp130 (Fig. 75.1).

Fig. 75.1 Localization of activated STAT3 (pSTAT3) 30 min after intravitreal injection of LIF. PBS injection did not induce detectable activation of STAT3 (b). While pSTAT3 was detected in all Müller cell nuclei in the

gp130f/f/VMD2-cre– retinas (c), apparent loss of STAT3 activation was observed in the gp130f/f/VMD2-cre+ retinas (arrowheads in d). Approximately 50% of Müller cell nuclei were no longer positive for pSTAT3. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Representative images were shown (n = 4)

75.3Effect of Impaired gp130 Activation in Müller Cells on LIF-Induced Photoreceptor Protection

Our previous study has demonstrated that activation of gp130 by LIF preserves photoreceptor function and prevents photoreceptor cell death from acute light damage (Ueki et al. 2008). The same acute light damage paradigm was used here to determine the role of Müller cell activation of gp130 in photoreceptor protection. Briefly, 6-week old gp130f/f/VMD2-cre+ mice were injected intravitreally with 0.5 μg/μl of LIF (1 μl volume) into the right eyes. Phosphate-buffered saline (PBS) was injected into the left eyes, serving as an internal control. gp130f/f/VMD2-cre- littermates were used as controls. Mice were then exposed to 3,000 lux of white fluorescent light for 4 hours (light damage) to determine the degree of gp130-induced photoreceptor protection. ERG and histological analyses were performed after 4 days of recovery under dim cyclic light.

The scotopic ERG a-waves from 8 animals per group were averaged at each flash intensity and were plotted in Fig. 75.2. In this light damage model, gp130f/f/VMD2cre- mice injected with PBS retained approximately 30% of the photoreceptor function of untreated mice (compare closed triangles and squares in Fig. 75.2). Eyes injected with LIF had significantly higher a-wave amplitudes than PBS-injected eyes (compare closed diamonds and triangles in Fig. 75.2), demonstrating LIFinduced protection of photoreceptor function. Interestingly, loss of gp130 activation in approximately 50% of Müller cells did not have any effect in protective ability of LIF (compare closed and open diamonds in Fig. 75.2).

Fig. 75.2 Scotopic a-wave amplitudes. ERGs were recorded from at least 8 mice per group and the average a-wave amplitudes were plotted versus the intensity of each light flash. Compared to the normal retina (uninjected/no LD; rectangles), PBS-injected control group with light exposure had approximately 70% decrease in the a-wave amplitude (triangles). LIF-injected group (diamonds) preserved the a-wave amplitude significantly after the light damage compared to PBS-injected group (triangles). Loss of gp130 activity in Müller cells did not affect the LIF-induced preservation of photoreceptor function from light damage (compare closed and open diamonds). Negative, gp130f/f/VMD2-cre– mice (closed symbols); positive, gp130f/f/VMD2-cre+ mice (open symbols); LD, light damage. Values are the means ± SEM

In order to determine the degree of light-induced cell death, eyes were collected and cross sections were obtained for histological evaluation (Fig. 75.3). Consistent with the a-wave amplitudes of scotopic ERG, loss of gp130 activation in Müller cells did not have any effect on ability of LIF to prevent photoreceptor cell death. The number of photoreceptors in gp130f/f/VMD2-cre+ retinas subjected to LIF injection followed by light damage was not different from that in gp130f/f/VMD2cre- controls (compare open and closed diamonds in Fig. 75.3b). Together, these results suggest that Müller cell activation of gp130 is not essential for photoreceptor protection.

75.4 Discussion

We have successfully demonstrated that approximately 50% of Müller cells in gp130f/f/VMD2-cre+ mice lost gp130-dependant activation of STAT3. Our data clearly demonstrate that this loss of gp130 activation in Müller cells does not impair LIF-induced photoreceptor protection from light damage. Many studies have

Fig. 75.3 LIF protects photoreceptors from light-induced cell death even after the loss of gp130 activity in Müller cells. (a) Representative histological sections through the superior retina near the optic nerve head are shown. (b) The number of photoreceptors lying in a single column (rows of photoreceptors) in the outer nuclear layer was counted along the vertical meridian of the eye at the optic nerve head. Untreated retinas are neither injected nor exposed to bright light. The number of photoreceptors present after light exposure was significantly higher in the LIF-injected groups (diamonds) compared to PBS-injected controls (triangles). Loss of gp130 activity in Müller cells did not affect the LIF-induced protection of photoreceptors from light-induced cell death (compare closed and open diamonds). Values are the means ± SEM (n = 8 per group)

focused on robust activation of signaling pathways in Müller cells immediately following the injection of gp130 ligands. Robust activation of ERK1/2 and STAT3 was detected in Müller cells in response to CNTF (Wahlin et al. 2000; Peterson et al. 2000; Wen et al. 2006) or LIF (Ueki et al. 2008), suggesting a possibility of indirect mechanisms of photoreceptor protection through Müller cell activation of gp130. On the other hand, our recent study demonstrated a possibility for direct mechanism of gp130-mediated photoreceptor protection through STAT3 activation in photoreceptors. While Müller cells are the first to respond to gp130 ligands, sustained activation of STAT3 was also detected progressively over time in all the retinal cells, including photoreceptors (Ueki et al. 2008). This delay in STAT3 activation in photoreceptors could be simply due to the rate of LIF diffusion through the retina. In addition, STAT3, but not ERK1/2 or Akt pathway, was active in the retina at the time of the